Molecular Determinants of Fibrillation in a Viral Amyloidogenic Domain from Combined Biochemical and Biophysical Studies
Abstract
:1. Introduction
2. Results and Discussion
2.1. Influence of pH on the Formation of HeV PNT3 Amyloid-like Fibrils
2.2. Rational Design and Generation of PNT3 Variants
2.2.1. Design of PNT3 Variants Targeting the EYYY Motif
2.2.2. Design of HeV PNT3 Truncated Variants Devoid of the C-Terminal Region
2.2.3. Design of a HeV PNT3 Variant Bearing a Unique Cysteine
2.2.4. Expression and Purification of the PNT3 Variants
2.2.5. Conformational Characterization of the PNT3 Variants
2.3. Relevance of the PNT3 EYYY Motif in Fibrillation Abilities
2.3.1. Aggregation Propensity of the EYYY Motif PNT3 Variants
2.3.2. Congo Red Binding Abilities of PNT3 EYYY Motif Variants
2.3.3. Propensity and Time-Dependance of Fibrillation of the PNT3 EYYY Motif Variants Using Negative-Staining Transmission Electron Microscopy (ns-TEM)
2.4. Impact of the HeV PNT3 C-Terminal Region in Fibrillation Abilities
2.4.1. Aggregation Propensity of C-Terminally Truncated PNT3 Variants
2.4.2. CR Binding Ability of C-Terminally Truncated PNT3 Variants
2.4.3. Propensity and Kinetics of Fibrillation of C-Terminally Truncated PNT3 Variants Using ns-TEM Studies
2.5. Impact of a Cysteine in the HeV PNT3 Sequence on Fibrillation Abilities
2.6. Characterization of the Aggregation Process by Taylor Dispersion Analysis
3. Materials and Methods
3.1. Generation of the Constructs
3.2. Proteins Expression and Purification
3.3. PEG Precipitation Assay (Relative Solubility)
3.4. Far-UV Circular Dichroism
3.5. Estimation of the Hydrodynamic Radius by SEC
3.6. Small-Angle X-ray Scattering (SAXS)
3.7. Congo Red Binding Assays
3.8. Negative-Staining Transmission Electron Microscopy (ns-TEM)
3.9. Kinetic Protein Aggregation Study by Taylor Dispersion Analysis (TDA)
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Proteins | Mass | RSOBS | RSNF | RSPMG | RSU | RSIDP | RSOBS/RSNF | RSOBS/RSPMG | RSOBS/RSU | RSOBS/RSIDP | CI |
---|---|---|---|---|---|---|---|---|---|---|---|
HeV PNT3 wt | 15198 | 27.0 ± 0.3 | 19.4 | 27.9 | 33.9 | 30.0 | 1.39 | 0.97 | 0.80 | 0.90 | 0.47 ± 0.02 |
HeV PNT33A | 14922 | 27.5 ± 0.4 | 19.3 | 27.7 | 33.5 | 30.0 | 1.42 | 0.99 | 0.82 | 0.92 | 0.42 ± 0.03 |
HeV PNT3A1 | 15106 | 28.4 ± 0.4 | 19.4 | 27.9 | 33.8 | 30.0 | 1.36 | 0.94 | 0.78 | 0.88 | 0.37 ± 0.03 |
HeV PNT3A2 | 15106 | 28.1 ± 0.3 | 19.4 | 27.9 | 33.8 | 30.0 | 1.31 | 0.91 | 0.76 | 0.85 | 0.39 ± 0.02 |
HeV PNT3A3 | 15106 | 27.5 ± 0.2 | 19.4 | 27.9 | 33.8 | 30.0 | 1.42 | 0.99 | 0.81 | 0.92 | 0.44 ± 0.01 |
NiV PNT3 | 14928 | 27.3 ± 0.7 | 19.3 | 27.7 | 33.5 | 30.5 | 1.41 | 0.99 | 0.82 | 0.90 | 0.44 ± 0.05 |
HeV PNT3 C-term_truncated | 9020 | 19.0 ± 0.1 | 16.1 | 22.6 | 25.8 | 22.7 | 1.17 | 0.84 | 0.73 | 0.83 | 0.71 ± 0.01 |
HeV PNT33A C-term_truncated | 8743 | 20.1 ± 0.6 | 16.0 | 22.4 | 25.4 | 22.7 | 1.26 | 0.90 | 0.79 | 0.89 | 0.56 ± 0.06 |
HeV PNT3_Cys | 15230 | 28.0 ± 0.4 | 19.5 | 28.0 | 33.9 | 30.0 | 1.44 | 1.00 | 0.83 | 0.93 | 0.41 ± 0.03 |
Proteins | I(0) cm−1 | Rg (Å) (Guinier) | Dmax (Å) | RgIDP (Å) | RgU (Å) |
---|---|---|---|---|---|
HeV PNT3 wt | 0.030 ± 1.8 × 10−4 | 36.67 ± 0.41 | 140 | 32.6 | 35.9 |
HeV PNT33A | 0.022 ± 8.1 × 10−5 | 39.48 ± 0.33 | 144 | 32.6 | 35.9 |
NiV PNT3 | 0.030 ± 8.2 × 10−5 | 37.37 ± 0.21 | 147 | 33.1 | 36.5 |
HeV PNT3 C-term_truncated | 0.018 ± 4.1 × 10−5 | 27.53 ± 0.14 | 115 | 24.5 | 25.9 |
HeV PNT33A C-term_truncated | 0.015 ± 5.6 × 10−5 | 27.34 ± 0.21 | 119 | 24.5 | 25.9 |
Instrument | SOLEIL Synchrotron (Gif-sur-Yvette, France) Beamline Swing |
---|---|
X-rays wavelength (Å) | 1.033 |
Energy (keV) | 12 |
Detector type | Dectris EIGER 4M |
Sample-to-detector distance (m) | 2.0 |
q-range | 0.003 − 0.549 Å−1 |
Temperature (°C) | 20 |
Samples | |
Concentration (mg mL−1) | 5 |
Sample volume (µL) | 50 |
Gel filtration column Flow rate (mL min−1) | AdvanceBio SEC 2.7 µm (Agilent) 0.3 |
Buffer | 50 mM sodium phosphate pH 7.2 (buffer C) |
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Nilsson, J.F.; Baroudi, H.; Gondelaud, F.; Pesce, G.; Bignon, C.; Ptchelkine, D.; Chamieh, J.; Cottet, H.; Kajava, A.V.; Longhi, S. Molecular Determinants of Fibrillation in a Viral Amyloidogenic Domain from Combined Biochemical and Biophysical Studies. Int. J. Mol. Sci. 2023, 24, 399. https://doi.org/10.3390/ijms24010399
Nilsson JF, Baroudi H, Gondelaud F, Pesce G, Bignon C, Ptchelkine D, Chamieh J, Cottet H, Kajava AV, Longhi S. Molecular Determinants of Fibrillation in a Viral Amyloidogenic Domain from Combined Biochemical and Biophysical Studies. International Journal of Molecular Sciences. 2023; 24(1):399. https://doi.org/10.3390/ijms24010399
Chicago/Turabian StyleNilsson, Juliet F., Hakima Baroudi, Frank Gondelaud, Giulia Pesce, Christophe Bignon, Denis Ptchelkine, Joseph Chamieh, Hervé Cottet, Andrey V. Kajava, and Sonia Longhi. 2023. "Molecular Determinants of Fibrillation in a Viral Amyloidogenic Domain from Combined Biochemical and Biophysical Studies" International Journal of Molecular Sciences 24, no. 1: 399. https://doi.org/10.3390/ijms24010399